RNA polymerase II-associating protein (RAP) 74 is the large subunit of human transcription factor (TF) IIF. This protein cooperates with TFIIA, TFIID, TFIIB, RNA polymerase II, RAP30, TFIIE, and TFIIH to accurately initiate transcription from a promoter. TFIIF (RAP30-RAP74) also stimulates the elongation rate of RNA polymerase II. RAP74 is phosphorylated in vivo on serine at unknown positions. Phosphorylation of RAP74 increases activity in accurate initiation and appears to strengthen the affinity of TFIIF for RNA polymerase II. We are interested in mapping the natural phosphorylation sites on RAP74 using mass spectrometry. Inspection of a serine-rich region within RAP74 revealed several potential casein kinase II sites. Casein kinase II phosphorylates RAP74 in vitro, and several of the sites that are modified have been identified using mass spectrometry. RAP74 also appears to be a substrate for phosphorylation by human transcription factors TAF II250 and TFIIH.